Abstract

Recent studies have associated osteopontin (OPN) with allergic inflammation; however, its role in human asthma remains unclear. The aim of this study was to measure OPN levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of healthy controls and asthmatics, identify cellular sources of OPN and examine possible correlations between OPN expression, disease severity and airway remodelling.

Serum samples were obtained from 35 mild-to-moderate asthmatics, 19 severe asthmatics and 17 healthy controls in the steady state and in cases of exacerbation. Of these subjects, 29 asthmatics and nine controls underwent bronchoscopy with endobronchial biopsy and BALF collection. OPN expression was determined by ELISA and immunohistochemistry/immunofluorescence. Reticular basement membrane thickness and goblet cell hyperplasia were also determined.

Serum and BALF OPN levels were significantly increased in all asthmatics in the steady state, whereas serum levels decreased during exacerbations. OPN was upregulated in the bronchial tissue of all patients, and expressed by epithelial, airway and vascular smooth muscle cells, myofibroblasts, T-lymphocytes and mast cells. OPN expression correlated with reticular basement membrane thickness and was more prominent in subepithelial inflammatory cells in severe compared to mild-to-moderate asthma.

OPN expression is upregulated in human asthma and associated with remodelling changes, and its subepithelial expression correlates with disease severity.

Asthma is an inflammatory disorder of the conducting airways, typically associated with aberrant type-2 T-helper cell (Th2) activation and response against environmental antigens. Airway inflammation in asthma is a multicellular process that is associated with structural alterations of the airway components, a process known as airway remodelling 1. The prominent role of airway inflammation and remodelling in the pathogenesis and clinical presentation of asthma has led to the current focus on mediators potentially involved in both processes.

Osteopontin (OPN) is a cytokine, originally described as a structural component of the extracellular matrix, that has the ability to bind to proteins and most types of collagen 2. OPN exists in a secreted form that mediates cell adhesion, migration and survival 3, and an intracellular nonsecreted form 4. OPN is produced by most cells of the immune system, including T-cells, B-cells, macrophages, neutrophils, eosinophils, natural killer cells and mast cells, as well as structural cells, including fibroblasts and smooth muscle and epithelial cells 5, 6. OPN has been typically classified as a type-1 cytokine 7. In humans, increased OPN expression has been observed in a number of Th1-mediated lung diseases, including granulomatous diseases and pulmonary fibrosis 8, 9.

There is now, also, emerging evidence to support an active role for OPN in Th2-linked inflammation and remodelling. OPN expression is upregulated in nasal tissue samples taken from asthmatic patients with chronic rhinosinusitis 10, and allergic patients undergoing successful long-term venom allergen immunotherapy show increased levels of serum OPN 11. Moreover, OPN is expressed and functional in peripheral blood eosinophils of atopic human subjects 12, and is also involved in the in vitro migration of eosinophils isolated from asthmatics 13. It has previously been demonstrated that OPN plays a crucial role in allergic airway inflammation by regulating distinct dendritic cell subsets 14, and that it is also involved in airway remodelling in well-established animal models of allergic airway disease 15. However, there are no studies investigating the expression pattern and cellular sources of OPN in patients with asthma of varying severity in a clinical setting. In the previous study 14, preliminary data were reported regarding OPN bronchial tissue expression in a limited number (six) of human asthmatics.

In the present study, it was hypothesised that the upregulation of OPN expression is more pronounced in more severe forms of asthma and that a relationship exists between OPN upregulation and remodelling changes. Furthermore, the aim was to identify the cellular sources of OPN in the bronchial tissue of asthmatics. Therefore, OPN expression was investigated in healthy controls and patients with varying severities of asthma in the steady state and in cases of exacerbation. Levels of OPN in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of mild-to-moderate (MMA) and severe asthma (SA) patients were determined and compared to those of healthy controls. It was also examined whether or not correlations exist between OPN expression, disease severity and control status, as well as tissue remodelling, with respect to reticular basement membrane (RBM) thickening and goblet cell hyperplasia. Finally, cellular sources of OPN were identified in the bronchial tissue of asthmatics using double immunofluorescence staining and confocal microscopy.

METHODS

Study population

A total of 54 persistently asthmatic patients (35 with MMA and 19 with SA) and 17 non-atopic healthy controls participated in the present study and were followed for ≥1 yr. From this population, 29 patients with steady-state asthma (12 MMA and 17 SA) and nine healthy controls consented to undergo research fibreoptic bronchoscopy with endobronchial biopsy and BALF collection when possible. Serum samples were collected from all participants in the steady state and the 17 patients that exhibited an exacerbation during the study. In addition, a subpopulation of 17 patients with MMA and SA agreed to receive 2 weeks of placebo-controlled treatment with oral corticosteroids (11 patients received 40 mg prednisolone and six received placebo; 2:1 ratio) in order to evaluate the effect of steroids on serum OPN expression. Informed and written consent was obtained from all participating subjects, and the corresponding protocol was approved by the Hospital Research Ethics Committee of Athens Chest Hospital, as well as the Greek National Organization for Medicines (both Athens, Greece). Subject characteristics are summarised in table 1, and patient groups and procedures are presented in a flow diagram (fig. 1).

Asthma was defined as a clear clinical history with current symptoms plus 15% reversibility in forced expiratory volume in 1 s (FEV1) after two puffs of β2-agonist and/or positive methacholine challenge. All patients had a physician-confirmed diagnosis of persistent asthma and were receiving therapy depending on their asthma severity, according to Global Initiative for Asthma guidelines 16. Asthma exacerbations were defined as events of severe deterioration of symptoms and rescue medication use that required the use of systemic corticosteroids, or an increase from a stable maintenance dose, for ≥3 days 17. All asthmatic patients had been attending the asthma outpatient clinic of Athens Chest Hospital for ≥2 yrs prior to the study. None of the patients were treated with theophylline, antileukotrienes or anti-immunoglobulin (Ig)E during the study. In addition, none of the participants had shown upper respiratory tract infection for 2 months prior to bronchoscopy or serum sample collection. Healthy individuals had no history of asthma or any other chronic disease, were not receiving any medication and gave normal spirometric results. All subjects participating in the study were nonsmokers. Atopy was defined according to the recently revised nomenclature of the World Allergy Organization 18, and was assessed by means of skin-prick tests for 18 common aeroallergens (HAL Allergy Benelux, Leiden, the Netherlands).

Pulmonary function testing

Pulmonary function tests were performed on the same day prior to bronchoscopy or on the day that serum samples were collected, as previously described 19.

Fibreoptic bronchoscopy

Bronchoscopy was performed on an outpatient basis at the Athens Chest Hospital according to published recommendations 20. All bronchoscopic procedures were performed by the same experienced bronchoscopist using a flexible bronchoscope (WM-N60 mobile workstation; Olympus, Tokyo, Japan). After inspection of the bronchial tree, a mini-lavage was performed according to the most recent European Respiratory Society Task Force guidelines 21. Briefly, 100 mL pre-warmed 0.9% saline were instilled into the right middle lobe in 20-mL aliquots and then gently aspirated. The first aliquot collected was discarded. Only BALF samples with a recovery rate of ≥60% were kept for further analysis, which was the case in six healthy controls and 17 patients with asthma. Endobronchial biopsy specimens were obtained following BALF collection, from various sites of the subsegmental carinae of the right lower lobes or right middle lobe. A minimum of three bronchial biopsy specimens were taken from each subject.

Sample collection and storage

Blood samples were centrifuged for 15 min at 1,000×g at room temperature. BALF samples were centrifuged for 15 min at 300×g at 4°C. Serum and BALF supernatant samples were aliquoted and stored at -80°C within 2 h after collection. BALF cell pellets were resuspended in 1 mL PBS and used for total and differential cell counts (table 2). Peripheral blood cell counts were also performed (table 3).

Table 3–Cell counts in the peripheral blood of healthy controls and asthmatic patients

ELISA

OPN concentration in serum and BALF was determined in duplicate using an ELISA kit (DY1433; R&D Systems, Wiesbaden, Germany) according to the manufacturer’s guidelines. Serum samples were diluted 50-fold before measurement. OPN levels in all samples were higher than the calculated detection limit of the assay (0.045 ng·mL−1). The intra- and inter-assay variability were determined to be 7.8 and 9.8%, respectively. The stability of the frozen serum and BALF samples was tested in 16 randomly selected samples from both patients (n = 10) and controls (n = 6), as previously described 19. The evaluation of the stability of OPN did not show significant differences among the four measurements performed in the serum (1st week: 39.93 (24.99–54.72) ng·mL−1, after 4 weeks: 41.61 (25.58–50.61) ng·mL−1, after 8 weeks: 40.59 (23.48–52.35) ng·mL−1, and after 12 weeks: 38.89 (23.35–49.75) ng·mL−1; p = 0.1) and BALF samples (1st week: 1.036 (0.939–1.329) ng·mL−1, after 4 weeks: 0.962 (0.785–1.312) ng·mL−1, after 8 weeks: 1.037 (0.698–1.357) ng·mL−1, and after 12 weeks: 0.9915 (0.755–1.279) ng·mL−1; p = 0.29).

Immunohistochemistry for OPN

Immunohistochemical staining for OPN was performed as previously described 14. Bronchial biopsy specimens were fixed in 10% neutral buffered formalin overnight and embedded in paraffin. Sections 3–4 μm thick were deparaffinised in xylol and rehydrated in a graded alcohol series. Endogenous peroxidase activity was inhibited using 0.3% H2O2 in methanol for 15 min. Heat-induced epitope retrieval was performed in pre-heated 10 mM citrate buffer (pH 6.0) using a microwave oven (600 W) for 15 min. Blocking of unspecific binding sites was performed by incubating slides with 10% goat serum for 30 min. Mouse anti-human monoclonal OPN antibody (diluted in PBS containing 1% bovine serum albumin BSA and 0.1% Triton X-100; MAB1433; R&D Systems; 1 μg·mL−1) was applied overnight at 4°C, followed by a 45-min incubation at room temperature with a biotinylated secondary goat anti-mouse antibody (BA-9200; Vector Laboratories, Burlingame, CA, USA). Slides were developed with the streptavidin–biotin complex–peroxidase reagent and the peroxidase substrate diaminobenzidine (DAB substrate kit (SK1400); Vector Laboratories) according to the manufacturer’s instructions. Negative controls were performed by substitution of the primary antibody with the same concentration of the corresponding isotypic IgG control (R&D Systems). All sections were counterstained with haematoxylin. All stained sections were coded and examined in a blinded manner at the end of the study. All available sections from each patient were evaluated. Biopsy specimens were considered suitable for examination when there was ≥1.0 mm of basement membrane length with intact epithelium and ≥0.1 mm2 of subepithelial area. At least three suitable biopsy sections were examined per subject.

Quantitative measurements of OPN-positive cells in the bronchial tissue were performed as previously described 14. Briefly, OPN-positive and -negative cells were counted in each biopsy specimen separately in the intact epithelium and the submucosa. Bronchial epithelial cells positive for the OPN monoclonal antibody were expressed as a percentage of total epithelial cells, whereas positive subepithelial inflammatory cells were expressed as a percentage of total cells. The intra-observer error was assessed by performing three separate counts of the same section on different occasions and was calculated to be <8%.

RBM thickness and goblet cell number measurements

Analysis of RBM thickness and subepithelial infiltration was performed on sections stained with haematoxylin–eosin as previously described 22. Briefly, basement membrane thickness was measured in micrometers by performing measurements at 20-μm intervals along the whole length of the membrane, taking only the adequately preserved and oriented area into account. A semi-quantitative scoring system, ranging 0–3, was used to grade the number of inflammatory cells infiltrating the subepithelial layer (table 2). Goblet cells were examined on lung sections stained with periodic acid–Schiff (395B-1KT; Sigma-Aldrich, Steinheim, Germany). The reaction of periodic acid selectively oxidises glucose residues and creates aldehydes that react with the Schiff reagent, staining carbohydrates in goblet cells a purple-magenta colour. Goblet cells were quantified as the number of positively staining cells per millimetre of basement membrane, as previously described 23. Sections were observed using an Axioskop40 microscopy system (Carl Zeiss Jena, Jena, Germany) coupled with an AxioCam MRc digital camera, and basement membrane thickness measurements were performed using AxioVision v4.5.0.0 image analysis software.

Confocal laser microscopy

Specimens that showed double staining on fluorescence microscopy were further examined using a confocal laser microscope. Fluorescence images of the tissue sections were acquired, using an Axiovert 200/LSM 510 META confocal microscopy system (Carl Zeiss Jena). DAPI was excited by the 405-nm laser diode and the fluorescence was collected using a long-pass (LP) 420 emission filter; the green (AF488) antibody was excited by the 488-nm argon ion laser line and the fluorescence was collected using a band-pass (BP) 505–530 emission filter, and the red (AF555) antibody was excited by the 543-nm green helium–neon laser line and the fluorescence was collected using an LP560 emission filter. Tissue sections were visualised using the confocal microscope at 1,024×1,024-pixel resolution through a Plan-Neofluar 40×/1.3 oil differential interference contrast objective with eight-times averaging in sequential scanning (multi-track) mode with the pinhole set to obtain an optical section of ∼1 μm in all channels.

Statistical analysis

Data are expressed as median with interquartile range (first and third quartiles) unless specified otherwise. Normality was assessed using Kolmogorov–Smirnov and D’Agostino–Pearson omnibus normality tests. Two-group comparisons were performed using the nonparametric Mann–Whitney U-test, whereas three-group comparisons were performed using the nonparametric Kruskal–Wallis one-way ANOVA accompanied by Dunn’s post hoc correction. Stability data were evaluated using the nonparametric Friedman test followed by Dunn’s post hoc analysis. The nonparametric Wilcoxon matched-pair test was applied to evaluate OPN expression in the stable state and during exacerbation. Correlation coefficients were calculated using the nonparametric Spearman’s rank method. A statistical software package was used for all data analysis and graph preparation (Prism v5; GraphPad, San Diego, CA, USA). A p-value of <0.05 was considered significant.

OPN in bronchial epithelial and subepithelial inflammatory cells in patients with asthma

Immunohistochemical staining revealed that OPN was highly expressed by both bronchial epithelial and subepithelial infiltrating inflammatory cells in asthmatic patients (fig. 3b and d), whereas healthy controls either did not express OPN or demonstrated very low OPN expression by some bronchial epithelial cells (fig. 3a). Detailed examination of the cellular sources of OPN in the lung revealed that OPN was expressed by cells positive for either α-SMA, CD3 or mast cell tryptase, suggesting that airway and vascular smooth muscle cells (fig. 4e–l), myofibroblasts (fig. 4m–p), T-lymphocytes (fig. 4q–t) and mast cells (fig. 4u–x) are sources of OPN production in the lung.

DISCUSSION

In the present study, we demonstrate that OPN is upregulated in bronchial tissue in asthma and show, for the first time, that subepithelial inflammatory cells in SA patients express significantly more OPN than in MMA patients, suggesting that OPN expression correlates with disease severity. Furthermore, we show that OPN expression correlates with RBM thickness, suggesting a role for OPN in asthma remodelling. We also demonstrate that OPN levels are increased in the serum of patients with asthma and that they decrease during exacerbations, and confirm that BALF OPN levels are elevated in asthma 12. Finally, our findings reveal, for the first time, that cellular sources of OPN in the bronchial tissue in asthma include not only epithelial cells and macrophages but also OPN localisation in airway and vascular smooth muscle cells, mast cells, lymphocytes and myofibroblasts.

The reasons behind the upregulation of OPN expression in asthma, as well as its role in the development of allergic inflammation and airway remodelling, remain largely unknown. OPN is generally classified as a pro-inflammatory cytokine 5, 13, 24, 25. However, emerging evidence supports an anti-inflammatory role of OPN in allergic disease. It has previously been shown that OPN has a protective role during secondary pulmonary antigenic challenge, and that therapeutic administration of recombinant OPN during secondary antigenic challenge protects from allergic disease 14. Furthermore, OPN appears to be associated with successful long-term venom allergen immunotherapy in allergic patients 11, it is involved in the prevention of viral infections 7 and may have a protective role in immune cell responses in the epithelium 26. Although it is possible that OPN upregulation in the bronchial tissue in asthma may initially serve as a protective mechanism, what has previously been demonstrated 14, 15 and further shown in the present study is that OPN overexpression in chronic inflammation may also result in tissue remodelling.

RBM thickness is a widely used marker of airway remodelling 1, and, in the present study, the RBM was significantly thicker in SA patients than in either MMA patients or healthy controls, in accordance with previous results 22. Interestingly, significant correlations were found between RBM thickness and bronchial tissue OPN expression, between FEV1 and RBM thickness, and between FEV1 and bronchial tissue OPN expression, suggesting that OPN is involved in remodelling and the resulting airway obstruction. Studies performed by our group have implicated OPN in fibrosis and remodelling in murine models of asthma 15. Moreover, OPN expression has recently been shown to be essential for the migration and differentiation of myofibroblasts 27, which are important contributors to RBM thickening. This is in accordance with the present findings, showing that myofibroblasts in the bronchial tissue of asthmatics express OPN in vivo. The relationship between OPN expression and subepithelial changes deserves further investigation, as it may suggest a target for therapeutic intervention.

Goblet cell hyperplasia and hypertrophy are also established pathological characteristics of asthma and airway remodelling 28. Using a murine model, it has previously been demonstrated that OPN is associated with increased mucus production 15; however, in the present study, no significant correlation was found. This could possibly be attributed to treatment, since steroids have been reported to ameliorate goblet cell hyperplasia and mucus production 29.

OPN levels in the serum of asthmatic patients in the steady state or during exacerbation have not previously been documented. Here, it is reported that serum OPN levels are increased in patients with asthma, possibly reflecting overspill from local infiltration and upregulation of OPN-producing cells in the bronchial tissue. However, although increased bronchial tissue OPN expression was found in SA, no differences were detected in the levels of OPN in either serum or BALF between MMA and SA patients. A hypothesis explaining this lack of difference could be that the higher bronchial OPN expression in SA occurs due to upregulation of the intracellular form of OPN. It was also found that serum OPN levels decrease during exacerbation; however, the reported significance is borderline and the number of cases is relatively small.

There is limited information regarding the cellular origin of OPN production in the bronchial airways of asthmatic patients. It has previously been reported that bronchial tissue macrophages from asthmatic patients can produce OPN 14, and, in the present study, it has been shown for the first time that OPN also localises in airway and vascular smooth muscle cells, mast cells, lymphocytes and myofibroblasts. This is in agreement with a previous report showing that OPN is expressed in circulating T-cells of subjects with atopic asthma 30, and that it is produced by mast cells and is involved in mast cell degranulation and migration 25. Furthermore, bronchial myofibroblasts from patients with usual interstitial pneumonia express OPN in vivo and in vitro31, and OPN expression is required for myofibroblast differentiation and activity 27. Lastly, OPN has previously been demonstrated to induce proliferation of human bronchial smooth muscle in vitro15.

There is currently no information regarding the effect of steroids on OPN expression in human lung. Moreover, the information available from animal models and human tissues other than lung is very limited and rather controversial 32, 33. In the present study, no differences were found in serum, BALF and bronchial tissue OPN expression between SA patients on long-term oral steroids and those on inhaled corticosteroids alone. Furthermore, no differences were found in serum OPN levels before and after 2 weeks of oral steroid treatment. In a recent study using a murine model of asthma, dexamethasone was found to decrease BALF OPN levels and lung tissue mRNA expression 34. However, the amount of steroids used in this study (3 mg·kg−1 dexamethasone or 20 mg·kg−1 prednisolone) is ∼200 times greater than the dose of oral prednisolone used in the present study, and 40 times higher than what current guidelines suggest for the management of severe exacerbations of asthma 16. It is, therefore, questionable whether the effects of dexamethasone on OPN expression reported in this experimental study are of clinical relevance.

The current study presents certain limitations. In an observational cross-sectional study, it is difficult to determine specific mechanisms of action. In addition, the number of patients that exhibited an exacerbation was relatively low, and the observed results could be subject to statistical error. This might also apply to BALF data; although 38 subjects underwent research bronchoscopy, BALF samples were processed from only 23 subjects in order to adhere to current guidelines regarding the recovery rate (>60%). In addition, it was not possible to differentiate between secreted and intracellular forms of OPN in the bronchial tissue. Finally, although a subgroup analysis was performed and no differences found in OPN expression with respect to steroid treatment, possible bias introduced by asthma medication cannot be excluded.

Interest regarding OPN has grown remarkably since the late 1990s, during which time OPN has progressed from a structural bone matrix component to an increasingly complex and multifaceted cytokine that has a pivotal role in the regulation of immune responses. In the present study, we demonstrate upregulation of OPN expression in human asthma that is associated with remodelling changes and correlates with disease severity. Further research on OPN function and regulation in human asthma could lead to novel therapeutic approaches targeting the regulation of OPN expression and function in situ.

Acknowledgments

The authors are grateful to all of the patients and healthy volunteers for their cooperation in the present study. The authors would also like to thank C. Malmhäll (Krefting Research Centre, Gothenburg, Sweden) for comments and advice on experiments and K. Papadopoulou (Pathology Dept, Athens Chest Hospital, Athens, Greece) for valuable technical assistance with the immunohistochemical procedures performed in this study. Finally, the authors also acknowledge the Centre for Cellular Imaging at the Sahlgrenska Academy (University of Gothenburg, Gothenburg, Sweden), and J. Fernandez-Rodriguez (Centre for Cellular Imaging) personally for comments and expert technical assistance with the confocal imaging equipment.

Footnotes

Support Statement

K. Samitas was funded by a European Academy of Allergy and Clinical Immunology (Zurich, Switzerland) fellowship. G. Xanthou and V. Panoutsakopoulou are supported by the Hellenic Ministries of Health (Athens, Greece) and Education (Marousi, Greece), and V. Panoutsakopoulou by a grant award (03ED750) from the General Secretariat for Research and Technology of the Hellenic Ministry of Development (Athens, Greece). A. Bossios and J. Lötvall are funded by the VBG GROUP Centre for Asthma and Allergy Research (Vänersborg, Sweden) and the Swedish Heart and Lung Foundation (Stockholm, Sweden; project No. 20070575).